C-31 desmethyl FR-900520 cyclic hemiketal immunosuppressant agent

ABSTRACT

Described is a process for producing a new immunosuppressant, a C-31 desmethyl, C-19/C-22 cyclic hemiketal biotransformation analog (Compound I) of FR-900520, under novel fermentation conditions utilizing the novel microorganism, Streptomyces, lavendulae ATCC No. 55209. Also disclosed is the C-31 methylated derivative (Compound II) of Compound I produced by enzymatic methylation using 31-O-desmethylimmunomycin O-methyl transferase, (DIMT), a methyl transferase enzyme. The macrolide immunosuppressants are useful in preventing human host rejection of foreign organ transplants, e.g. bone marrow, liver, lung, kidney and heart transplants.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a new FK-506 type immunosuppressant agent, aC-31 desmethyl, C-19/C-22 cyclic hemiketal analog of FR-900520 (FK-520),Compound I, a novel fermentation process for its production, utilizingthe novel microorganism Streptomyces lavendulae ATCC No. 55209. Theprocess involves culturing the microorganism in the presence ofFR-900520 under conditions which induce C-31 demethylation and C-19/C-22cyclic hemiketal formation of FR-900520. Also disclosed is the C-31methylated derivative of Compound I, designated as Compound II, producedby enzymatic methylation of Compound I using DIMT, a methyl transferaseenzyme. Also disclosed is a method of its use in a human host fortreatment of autoimmune diseases, infectious diseases and/or preventionof organ transplant rejections.

2. Brief Description of Disclosures in the Art

In 1983, the U.S. FDA approved cyclosporin, and extremely effectiveanti-rejection drug that revolutionized the field of organ transplantsurgery. The drug acts by inhibiting the body's immune system frommobilizing its vast arsenal of natural protecting agents to reject thetransplant's foreign protein.

As effective as the drug is in fighting transplantation rejection, itsuffers drawbacks in causing kidney failure, liver damage and ulcerswhich in many cases can be very severe.

EPO Publication No. 0184162 to Fujisawa, hereby incorporated byreference, describes a new macrolide immunosuppressant FK-506 which isreputed to be 100 times more effective than cyclosporin. The macrolideis produced by fermentation of a particular strain of Streptomycestsukubaensis. Also described is the closely related macrolideimmunosuppressant FR-900520, produced by S. hygroscopicus subsp.yakushimaensis. The terms "FK-520" and "immunomycin" also used by Merck& Co. Inc. as synonyms for FR-900520.

U.S. Pat. No. 3,244,592 to T. Arai describes the culturing ofStreptomyces hygroscopicus var. ascomyceticus to produce the antifungal"ascomycin", which has been shown to be the same compound as FR-900520.

There is, however, no description in the literature of the production ofany FK-506 type immunosuppressive agents, which substantially lack theside effects or similar side effects to cyclosporin.

In this regard, new FK-506 type immunosuppressants are continuouslybeing searched for.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is an ¹ H nuclear magnetic resonance (NMR) spectrum taken at 400MHz of Compound I in CDCl₃, and FIG. 1B illustrates the assignedmolecular structure.

FIG. 2A is an ¹ H MR spectrum taken at 400 MHz of Compound II in CDCl₃solution and FIG. 2B illustrates the assigned molecular structure.

FIG. 3A is a ¹ H NMR spectrum taken at 400 MHz in CDCl₃ of FR-90052,also designated as "immunomycin" and FIG. 3B illustrates its assignedmolecular structure.

SUMMARY OF THE INVENTION

It has been found that a new FK-506 immunosuppressant, Compound I, canbe obtained by the fermentation of the microorganism Streptomyceslavendulae ATCC No. 55209, together with the macrolide immunosuppressantFR-900520, under submerged aerobic conditions in an aqueous carbohydratemedium, containing a nitrogen nutrient, said conditions being conductedat a pH of about 7, which are sufficient to C-31 demethylate FR-900520and induce C-19/C-22 cyclic hemiketal formation.

The resultant Compound I exhibits FK-506 immunosuppressant activity,i.e., positive inhibition of T-cell activation, as demonstrated by thecalcium ionophore (ionomycin) plus phorbol myristate acetate (PMA)induced T-cell stimulation assay, also referred to herein as the "T-cellproliferation assay".

The principle of this assay, as described in J. Immunology, Vol. 144,pp. 251-258 (1990) by F. Dumont et al, is to measure the proliferationof mouse T lymphocytes stimulated with the combination of ionomycin plusPMA. A positive sample e.g. FK-506, in this assay will inhibit T-cellproliferation, as indicated by reduced tritiated thymidine uptake.

Also disclosed is the C-31 methylated analog of Compound I, designatedas Compound II, produced by enzymatic methylation of Compound I by DIMT,a methyl transferase enzyme.

In accordance with this invention, there is provided a process forproducing a new FK-506 immunosuppressant, identified as Compound I,comprising the step of culturing a strain of Streptomyces lavendulae,ATCC No. 55209, together with FR-900520 under submerged aerobicfermentation conditions in an aqueous carbohydrate medium, containing anitrogen nutrient, for a sufficient time to produce product Compound I.

Further provided is a new FK-506 immunosuppressant, Compound I, producedby the above process which exhibits positive inhibition of T-cellactivation by the T-cell proliferation assay and exhibits a protonnuclear magnetic resonance spectrum illustrated in FIG. 1 and has anassigned structure as also identified in FIG. 1.

Also provided is a pharmaceutical composition containing atherapeutically effective amount of Compound I in combination with apharmaceutically acceptable, substantially non-toxic carrier orexcipient.

In addition there is provided a new FK-506 immunosuppressant, CompoundII, which exhibits positive inhibition of T-cell activation by theT-cell proliferation assay and exhibits a proton nuclear magneticresonance spectrum illustrated in FIG. 2 and has an assigned structureas also identified in FIG. 2.

Also provided is a pharmaceutical composition containing atherapeutically effective amount of Compound II in combination with apharmaceutically acceptable, substantially non-toxic carriere orexcipient.

In addition, there is provided a method of use for treating human hostto prevent transplantation rejection, or for treating autoimmune diseaseor infectious disease comprising administering to said host atherapeutically effective amount of Compound I or II.

Furthermore there is provided a biologically pure culture ofStreptomyces lavendulae, ATCC No. 55209.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

The present invention involves the fermentation of Streptomyceslavendulae, ATCC No. 55209, together with FR-900520 to produce CompoundI. The micro-organism is currently on deposit under the terms of theBudapest Treaty with the American Type Culture Collection, 12301Parklawn Dr. in Rockville, Md. as ATCC No. 55209. The microorganism isalso maintained in The Merck Culture Collection as MA 6954. The physicalcharacteristics and taxonomy, including morphological, cultural,biological and physiological characteristics are briefly describedhereinbelow.

The following is a general description of Streptomyces lavendulae strainATCC 55209 which transforms immunoregulant FR-900520 to Compound I.Observations of growth, general cultural characteristics and carbonsource utilization were made in accordance with the methods of Shirlingand Gottleib (Internat. J. System. Bacteriol. 16:313-340). Chemicalcomposition of the cells was determined using the methods of Lechevalierand Lechevalier (in Actinomycete Taxonomy, A. Dietz and D. W. Thayer,Ed. Society for Industrial Microbiology, 1980). Coloration of theculture was determined by comparison with color standards contained inthe Inter-Society Color Council-National Bureau of Standards CentroidColor Charts (U.S. Dept. of Commerce National Bureau of StandardsSupplement to NBS Circular 553, 1985).

Source--This culture was isolated from a soil sample collected inPiscataway, N.J.

Analyst Wall Composition--Peptidoglycan contains LL-diaminopimelic acid.

General growth characteristics--Good growth on yeast malt extract agar(YME), inorganic salt starch agar, oatmeal, trypticase soy agar, andpeptone iron agar. Moderate growth on glycerol asparagine agar. Sparsegrowth on Czapek's agar and tap water agar supplemented with NZ-amine(Shefield Chemical Co.) Culture also grows in tryptone yeast extractbroth. Culture grows at 27° C. and 37° C.

Colony morpholgy--(on YME at 21 d) Substrate mycelium is slightly yellowbrown. Aerial mycelium white. Spore mass is abundant and pinkish gray incolor. Colonies are opaque, raised, with lobate edges, rubbery intexture with a rough surface texture.

Micromorphology--Aerial mycelia (0.76 μm) arise from substrate myceliaand are branched and slightly flexous. In mature cultures (7-28d p.i.)the aerial mycelium terminates in flexous chains of spores thatoccasionally terminate in hooks, loops or extended spirals. Thischaracteristic is especially noticeable in areas of dense aerialdevelopment. Sporulation occurs on YME, inorganic salts-starch agar,gylcerol asparagine agar, tap water agar with NZ-amine and Czapek'sagar.

Miscell physiological reactions Culture--produces H₂ S in peptone-ironagar. Melanoid pigments are formed in TY broth and on peptone iron agarslants. Starch is weakly hydrolyzed. Carbon source utilization patternis as follows: moderate utilization of cellobiose, α-D-glucose,D-maltose, and D-mannose; poor utilization of D-fructose, α-D lactose,β-D-lactose; no utilization of D-arabinose, L-arabinose, inositol,D-mannitol, D-raffinose, L-rhamnose, sucrose, D-xylose, or L-xylose.

Diaznosis--Cell wall analysis reveals that ATCC #55209 has a type I cellwall. Morphological studies reveal that the culture produces long chainsof spores on flexous sporophores that occasionally terminate in loops,hooks, or extended spirals. Sporophores arise from the aerial mycelium.These are characteristics typical for strains of Streptomyces.

A comparison of the phenotypic data of ATCC No. 55209 with that of thevalidly published species of Streptomyces in the taxonomic literature(following References 1-7) shows that this strain strongly resemblesStreptomyces lavendulae, Streptomyces virziniae, Streptomycesflavotricini, Streptomyces goshikiensis, and Streptomyces colombiensis.These species fail into either the gray or red series andcharacteristically produce spores on flexous sporophores that terminatein loops, hooks or spirals. Most produce melanoid pigments and allessentially exhibit identical carbon source utilization patterns.Streptomyces virginiae, Streptomyces flavotricini, Streptomycesgoshkiensis, and Streptomyces colombiensis are currently considered tobe subjective synonyms of Streptomyces lavendulae (7). Based upon theseresults, ATCC No. 52209 is considered to be a novel strain ofStreptomyces lavendulae.

Literature References

1. Shirling, E. B. and Gottlieb, D., Int. J. System Bacteriol.18:69(1968)

2. Shirling, E. B. and Gottlieb, D., Int. J. System, Bacteriol.18:279(1968)

3. Shirling, E. B. and Gottlieb, D., Int. J. System, Bacteriol.19:391(1969)

4. Shirling, E. B. and Gottlieb, D., Int. J. System, Bacteriol.22:265(1972)

5. Nonomura, H. J., Ferment. Technol.52:78 (1974)

6. Pridham, T. and Tresner, H., in Bergey's Manual of DeterminativeBacteriology, Eighth Edition, R. E. Buchanan and N. E. Gibbons, Ed.,Williams and Wilkins, Baltimore (1974)

7. Loci, R. in Bergy's Manual of Systematic Bacteriology, Vol.4., St.Williams, M. E. Sharpe and J. G. Holt. Ed., Williams and Wilkins,Baltimore. (1989)

    ______________________________________                                        Carbohydrate utilization patterns of                                          ATCC 55209 at 21 day                                                          Carbon source  Utilization by ATCC 55209                                      ______________________________________                                        D-arabinose    0                                                              L-arabinose    0                                                              cellobiose     2                                                              D-fructose     1                                                              inositol       0                                                              α-D-lactose                                                                            1                                                              β-D-lactose                                                                             1                                                              D-maltose      2                                                              D-mannitol     0                                                              D-mannose      2                                                              D-raffinose    0                                                              L-rhamnose     0                                                              sucrose        0                                                              D-xylose       0                                                              L-xylose       0                                                              a-D-glucose (control)                                                                        2                                                              ______________________________________                                         3 = good utilization,                                                         2 = moderate utilization,                                                     1 = poor utilization,                                                         0 = no utilization                                                       

    ______________________________________                                        Cultural characteristics of ATCC 55209 at 21 days                                     Amount of                                                                     Growth                                                                        ATCC      Aerial Mycelium                                                                            Soluble                                                                              Reverse                                 Medium  55029     ATCC 55029   Pigments                                                                             Color                                   ______________________________________                                        Yeast   Good      Aerial mycelium                                                                            none   Slight                                  Extract           pinkish gray noted  yellow                                  Malt              (10 pk Gray).       brown                                   Extract           Spores borne in     (74 s.                                                    slightly flexous    yBr)                                                      chains with ter-                                                              minal hooks and                                                               loops.                                                      Gluocose                                                                              Moderate  Aerial mycelium                                                                            none   Yellow                                  Asparagine        pinkish gray (10)                                                                          noted  white                                                     pk Gray). Spores    (92 y.                                                    borne in slightly   White)                                                    flexous chains                                                                with occasional                                                               hooks and loops.                                            Inorganic                                                                             Good      Aerial mycelium                                                                            none   Yellow                                  Salts             pinkish gray (10)                                                                          noted  white                                   Starch            pk Gray). Spores    (92 y.                                                    borne in slightly   White)                                                    flexous chains                                                                with numerous                                                                 hooks and loops.                                                              Starch weakly                                                                 hydrolyzed.                                                 Oatmeal Good      Aerial mycelium                                                                            none   Yellow                                                    pinkish gray (10)                                                                          noted  white                                                     pk Gray). Spores    (92 y.                                                    borne in slightly   White)                                                    flexous chains                                                                with hooks and                                                                loops. Starch                                                                 weakly hydro-                                                                 lyzed.                                                      Tap Water                                                                             Sparse    Yellow white none   Yellow                                                    (92 y, White).                                                                             noted  white                                                     Spores borne in     (92 y.                                                    slightly flexous    White)                                                    chains with hooks,                                                            loops and spirals.                                          Czpak   Sparse    Yellow white none   Yellow                                                    (92 y, White).                                                                             noted  white                                                     Flexous aerial      (92 y,                                                    mycelium.           White)                                  Peptone Good        --         Melanin                                                                               --                                     Iron                           positive,                                                                     H.sub.2 S                                                                     positive.                                      ______________________________________                                    

The present invention process can be practiced with any Compound Iproducing strain of Streptomyces lavendulae, and particularly preferredis the ATCC No. 55209 strain.

In general, Compound I can be produced by culturing (fermentation) ofthe Actinomycete strain, in an aqueous nutrient medium containingsources of assimilable carbon and nitrogen, preferably under submergedaerobic conditions (e.g. shaking culture, submerged culture, etc.). Theaqueous medium is preferably maintained at a pH of about 7 at theinitiation and termination (harvest) of the fermentation process. Ahigher pH leads to substantial and/or total loss of product. The desiredpH may be maintained by the use of a buffer such asmorpholinoethanesulfonic acid (MES), morpholinopropanesulfonic acid(MOPS), and the like, or by choice of nutrient materials whichinherently possess buffering properties, such as production mediadescribed hereinbelow.

The preferred sources of carbon in the nutrient medium are carbohydratessuch as glucose, xylose, galactose, glycerin, starch, dextrin, and thelike. Other sources which may be included are maltose, raffinose,mannose, salicin, sodium succinate, and the like.

The preferred sources of nitrogen are yeast extract, meat extract,peptone, gluten meal, cottonseed meal, soybean meal and other vegetablemeals (partially or totally defatted), casein hydrolysates, soybeanhydrolysates and yeast hydrolysates, corn steep liquor, dried yeast,wheat germ, feather meal, peanut powder, distiller's solubles, etc., aswell as inorganic and organic nitrogen compounds such as ammonium salts(e.g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.),urea, amino acids, and the like.

The carbon and nitrogen sources, though advantageously employed incombination, need not be used in their pure form, because less purematerials which contain traces of growth factors and considerablequantities of mineral nutrients, are also suitable for use. Whendesired, there may be added to the medium mineral salts such as sodiumor calcium carbonate, sodium or potassium phosphate, sodium or potassiumchloride, sodium or potassium iodide, magnesium salts, copper salts,cobalt salts, and the like. If necessary, especially when the culturemedium foams seriously, a defoaming agent, such as liquid paraffin,fatty oil, plant oil, polypropylene glycol, mineral oil or silicone maybe added.

The FR-900520 (also referred to as "FK-520" by Merck) starting materialcan be obtained by the fermentation of S. hygroscopicus var.ascomyceticus, ATCC No. 14891, as described in U.S. Pat. No. 3,244,592,and by the fermentation of S. hygroscopicus subsp. yakushimaensis No.7278, to produce FR-900520, as described in EPO Publication No. 0184162to Fujisawa, and U.S. Pat. No. 4,894,366.

As to the conditions for the production of Compound I in massiveamounts, submerged aerobic cultural conditions are preferred therefor.For the production in small amounts, a shaking or surface culture in aflask or bottle is employed. Furthermore, when the growth is carried outin large tanks, it is preferable to use the vegetative form of theorganism for inoculation in the production tanks in order to avoidgrowth lag in the process of production of Compound I. Accordingly, itis desirable first to produce a vegetative inoculum of the organism byinoculating a relatively small quantity of culture medium with spores ormycelia of the organism produced in a "slant" and culturing saidinoculated medium, also called the "seed medium", and then to transferthe cultured vegetative inoculum aseptically to large tanks. Thefermentation medium, in which the inoculum is produced, is substantiallythe same as or different from the medium utilized for the production ofCompound I and is generally autoclaved to sterilize the medium prior toinoculation. The pH of the medium is generally adjusted to about 7.0prior to the autoclaving step by suitable addition of an acid or base,preferably in the form of a buffering solution.

Agitation and aeration of the culture mixture may be accomplished in avariety of ways. Agitation may be provided by a propeller or similarmechanical agitation equipment, by revolving or shaking the fermentor,by various pumping equipment or by the passage of sterile air throughthe medium. Aeration may be effected by passing sterile air through thefermentation mixture.

The fermentation is usually conducted at a temperature between about 20°C. and 40° C., preferably 25°-35° C., for a period of about 10 hours to20 hours, which may be varied according to fermentation conditions andscales. Preferably, the production cultures are incubated for about 17hours at 27° C. on a rotary shaker operating at 220 rpm, wherein the pHof the fermentation medium is maintained at 7.0 to harvest.

Preferred culturing/production media for carrying out the fermentationinclude the following media:

    ______________________________________                                        Seed Medium         g/liter                                                   ______________________________________                                        Dextrose            1.0                                                       Dextrin             10.0                                                      Beef Extract        3.0                                                       Ardamine pH         5.0                                                       NZ Amine Type E     5.0                                                       MgSO.sub.4.7H.sub.2 O                                                                             0.05                                                      K.sub.2 HPO.sub.4   0.37                                                      Adjust pH to 7.1                                                              Add CaCO.sub.3 0.5 g/l                                                        ______________________________________                                        Transformation Medium B                                                                           g/liter                                                   ______________________________________                                        Glucose             20                                                        Soya Meal           5                                                         Yeast Autolysate    5                                                         NaCI                5                                                         MES                 9.8                                                       Adjust pH to 7.0                                                              ______________________________________                                    

The produced Compound I can be recovered from the culture medium byconventional means which are commonly used for the recovery of otherknown biologically active substances. The Compound I substance producedis found in the cultured mycelium and filtrate, and accordingly can beisolated and purified from the mycelium and the filtrate, which areobtained by filtering or centrifuging the cultured broth, by aconventional method such as concentration under reduced pressure,lyophilization, extraction with a conventional solvent, such as methanoland the like, pH adjustment, treatment with a conventional resin (e.g.anion or cation exchange resin, non-ionic adsorption resin, etc.),treatment with a conventional adsorbent (e.g. activated charcoal,silicic acid, silica gel, cellulose, alumina, etc.), crystallization,recrystallization, and the like. A preferred method is solventextraction, particularly using methanol.

The product Compound I from the fermentation exhibits antagonistactivity versus the FK-506 immunosuppressive activity by the "T-cellproliferation assay" and possesses utility on this basis and exhibitsthe following physical characteristics:

1. White amorphous powder

2. Solubility in methanol

3. Molecular weight of 793, as determined by FAB mass spectroscopy(observed M+Li=800), is consistent with the assigned structure in FIG.1.

The Compound I obtained according to the fermentation processes asexplained above can be isolated and purified in a conventional manner,for example, extraction, precipitation, fractional crystallization,recrystallization, chromatography, and the like.

It is to be noted that in the aforementioned fermentation reactions andthe post-treatment of the fermentation mixture therein, the conformerand/or stereo isomer(s) of Compound I due to asymmetric carbon atom(s)or double bond(s) of the Compound I may occasionally be transformed intothe other conformer and/or stereoisomer(s), and such cases are alsoincluded within the scope of the present invention.

Also described is a new enzyme, 31-O-desmethyl-immunomycinO-methyltransferase (DIMT) which can specifically and preferentiallymethylate the C-31 hydroxy group in Compound I to produce Compound II.The enzyme can be extracted out of the microorganism Streptomyceshygroscopicus var. ascomyceticus, ATCC No. 55087. Employing the enzymein its active form, supplemented with Mg⁺² ion, in the presence of themethyl donor, S-adenosyl methionine (SAM), Compound I can be C-31methylated to produce Compound II.

The enzyme described can be used as a cell free or purified enzyme.Purified DIMT has a molecular weight of about 32 K daltons, as measuredby SDS-PAGE, an isoelectric point (PI) of 4.4, and capable of catalyzingthe C-31 O-methylaion of a C-31 hydroxy containing FK-506 type molecule,in the presence of a methyl transfer agent, when supplemented with Mg⁺²ion.

The enzyme can be isolated from the microorganism, Streptomyceshygroscopicus var. ascomyceticus, ATCC No. 55087 and purified forenzymatic use by the procedure described in the Examples.

The enzyme is useful in a process for methylating the C-31 hydroxy groupin Compound I involving the step of contacting Compound I with themethyl transfer agent, e.g., S-adenosyl methionine, in the presence ofthe DIMT enzyme described, supplemented with Mg⁺² ion, in an aqueoussolvent therefor.

Generally, the process is conducted at a pH from about 7-9 and in thetemperature range of about 25°-40° C.

The aqueous solvent system is generally a phosphate buffer of aboutpH=7-8.

The Mg⁺² ion is supplied as a soluble magnesium salt, e.g., magnesiumchloride, magnesium sulfate, magnesium citrate, and the like.

Isolation and purification of Compound II can be accomplished byconventional techniques including HPLC or reverse phase HPLC asdescribed in the examples.

The enzyme is specific in methylating only the C-31 hydroxy group inCompound I.

Structures of the compounds involved in this invention include thefollowing: ##STR1##

The FR-900520 immunosuppressant is disclosed by Fujisawa in EPO 0184162,having a slightly different numbering system than Merck, i.e. ##STR2##with the assigned chemical name;17-Ethyl-1,14-dihydroxy-12-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylvinyl]-23-25-dimethoxy-13,19,21,27-tetramethyl-11,28-dioxa-4-azatricyclo[22.3.1.0⁴,9 ]octacos-18-ene-2,3,10,16-tetraone.

The Compounds I and II of the present invention possessimmunosuppressant pharmacological activity therefore are useful in atherapeutic program involving FK-506 type therapy designed for thetreatment and prevention of the transplantation rejection of organs ortissues such as heart, kidney, liver, medulla ossium, skin, etc.,graft-versus-host diseases by medulla ossium transplantation, autoimmunediseases such as rheumatoid arthritis, systemic lupus erythematosus,Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type Idiabetes, uveitis, and the like.

The pharmaceutical compositions of this invention can be used in theform of a pharmaceutical preparation, for example, in solid, semisolidor liquid form, which contain the Compounds I or II, of the presentinvention, as an active ingredient, in admixture with an organic orinorganic carrier orexcipient suitable for external, enteral orparenteral applications. The active ingredient may be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use. The carriers which canbe used are water, glucose, lactose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form,and in addition auxiliary, stabilizing, thickening and coloring agentsand perfumes may be used. The active object compound is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the process or condition of diseases.

For applying this composition to a human, it is preferable to apply itby parenteral or enteral administration. While the dosage oftherapeutically effective amount of the Compound I or II, varies from,and also depends upon the age and condition of each individual patientto be treated, a daily dose (calculated on the basis of a 70 kg man) ofabout 0.01-1000 mg, preferably 0.1-500 mg and more preferably 0.5-100mg, of the active ingredient is generally given for treating diseases,and an average single dose of about 0.5 mg, 1 mg, 5 mg, 10 mg, 50 mg,100 mg, 250 mg and 500 mg is generally administered.

The following examples are given for the purpose of illustrating thepresent invention and should not be construed as being limitations onthe scope or spirit of the instant invention.

EXAMPLE 1 Microorganism and Culture Conditions

A frozen vial of culture, ATCC No. 55209, was used to inoculate a 250 mlbaffled shake flask containing 50 ml of an autoclaved (sterilized) seedmedium consisting of (in units of grams/liter) dextrin 10.0, dextrose1.0, beef extract 3.0, ardamine PH (Yeast Products, Inc.) 5.0, N-Z Aminetype E 5.0, MgSO₄.7H₂ O 0.05, KH₂ PO₄ 0.37, and CaCO₃ 0.5. The pH of theseed medium was adjusted to 7.1 before autoclaving. The seed wasincubated in the seed medium at 27° C. for 24 hours on a rotary shakeroperating at 220 rpm. A 2.5 ml aliquot of the resulting seed medium wasused to inoculate a 250 ml non-baffled shake flask containing 50 ml ofthe following previously autoclaved (sterilized) fermentation medium:

    ______________________________________                                        Soy-Glucose Medium                                                            ______________________________________                                        Glucose            20.0                                                       Soya Meal          5.0                                                        Yeast Autolysate   5.0                                                        NaCl               5.0                                                        MES                9.8                                                        AdJust pH to 7.0                                                              ______________________________________                                    

FR-900520 was added as a solution in dimethylsulfoxide to achieve afinal concentration of 0.05 mg/ml concentration. The shake flaskcontents were subsequently incubated for 48 hrs. at 27° C. on a rotaryshaker operating at 220 rpm.

Isolation and Purification

The whole broth (250 ml) was extracted three times with methylenechloride (3×250 ml). Methylene chloride extracts were combined, driedover sodium sulfate, and concentrated under vacuum to an oily residue.The residue was dissolved in methanol and subjected to high performanceliquid chromatography (HPLC). HPLC was carried out on Whatman Magnum 20Partisil 10 ODS-3 Column (22.1 mm ID×25 cm) at room temperature andmonitored at 205 nm. The column was developed at 7 ml/min with a 65minutes linear gradient from 35% to 80% acetonitrile in 0.1% phosphoricacid. The compounds were collected during repeated injections of theabove described extract. The fraction with a retention time of 57minutes (Compound I) was pooled, adjusted to pH 4.0, evaporated toremove acetonitrile, and desalted using a C18 Sep Pak (Waters Associate)to yield 4 mg of Compound I.

ANALYTICAL SPECTRAL DATA

Mass spectral data of the subject Compound I biotransformation productobtained by the incubation of FR-900520 with culture ATCC #55209,coupled with the NMR data, as shown in the spectrum of FIG. 1, isconsistent with the following assigned molecular structured (I), alsoshown in FIG. 1 and illustrates the C-31 desmethyl and C-19/C-22 cyclichemiketal characteristics in the structure: ##STR3## Key features of theNMR analysis of the above biotransformation product which are consistentwith the 31-desmethyl cyclized structure (I) are given by the followingpoints:

1. Loss of methoxy assigned to the 31-OCH₃ based on the downfield shiftof H-31;

2. Absence of the 19-methyl;

3. Presence of a novel CH₂ O (3.74 ppm and 4.06 ppm, Jgem:16 Hz)reasonably assigned to a derivatized 19-methyl;

4. Absence of typical 23-methylene signals;

5. Upfield shifted H-21;

6. A 0.6 ppm downfield displacement of H-20 and reduction of its vicinalcoupling constant from 9 to approximately 7 Hz.

Reduction of the 22 ketone is implied by the above points 4 and 5. Whilethe proton NMR spectrum did not allow a decision between a 19-CH20H anda 19-CH20R, the perturbations of H-20 (point 6) were suggestive of acyclic structure. Based on a consideration of several possiblitites, I,is entirely consistent with both NMR and MS observations (i.e., theincrease of two mass units is accounted for by the loss of methoxyl andthe incorporation of oxygen).

EXAMPLE 2 Isolation and characterization of S-adenosyl-L-methionine:31-O-desmethylimmunomycin O-methyl-transferase

Immunomycin (See Structure, supra) is a new macrocylic lactone/lactamantibiotic with effective immunosuppressive and antifungal activities(See EP Publication No. 0323865 (1989) by F. J. Dumont, et al.).Precursor feeding studies in this laboratory has established thepolyketide nature of the macrocyclic structure. Furthermore, the datafrom the feeding studies indicate that the three methyl groups atpositions 13, 15 and 31 originate from methionine throughS-adenosyl-L-methionine (SAM). With the development of a mutant strainof S. hygroscopicus var. ascomyceticus (MA 6674) which produces31-O-desmeihylimmunonycin, it has become possible to isolate andcharacterize the enzyme, S-adenosyl-L-methionine:31-O-desmethylimmunomycin O-methyltransferase (DIMT).

Bacterial Growth and Mycelium Preparation

Frozen vegetative mycelium of Streptomyces hygroscoipicus var.ascomyceticus (ATCC 55087) was cultured in the seed and fermentationmedia. Mycelial cells were harvested at 72 hours and washed three timeswith 50mm phosphate buffer, pH 7.5, containing 0.5M KCl and finally withthe same buffer with no KCl. Washed mycelium was used for thepreparation of the cell-free extract.

Time Course of the Mycelial Growth and Immunomycin Production

At different time intervals, an aliquot of the fermentation culture wascentrifuged at 2000 rpm for 10 minutes and packed cell volume wasdetermined. Similarly, an aliquot was extracted with methanol andimmunomycin production was quantitated by HPLC.

S-adenosyl-L-methionine: 31-O-desmethylimmunomycin O-methyltransferase(DIMT) Assay and Product Identification

The assay was carried out in 1 ml. mixture containing 0.025 mM31-desmethylimmunomycin, 1 mM MgSO₄ and different quantities of theenzyme source in 50 mM phosphate buffer, pH 7.5. Reaction was initiatedby the addition of 1 nmole of ¹⁴ C-SAM (S-adenosyl-L-[methyl-¹⁴ C]methionine with the specific activity of 46 mCi/mmole. Incubation of thecomplete mixture was carried out at 34° C. for 20 minutes and thereaction was terminated by the addition of ethyl acetate. Product of thereaction was extracted with 2 ml of ethyl acetate and 1 ml of theextract was used for TLC and HPLC analysis. For the analysis of theradioactive reaction product (immunomycin), the ethyl acetate extractwas spiked with the standard immunomycin and subjected to the TLC with aplate having plastic support. The plastic sheet was developed inchloroform:methanol (9:1) and area showing standard immunomycin underUV-light was cut and the radioactivity in the cut-strip was measured.The amount of the product formed was calculated from the measuredradioactivity. For the HPLC analysis of the product of the enzymereaction, several enzymatic reaction mixtures were pooled and theextract was subjected to the TLC analysis as above. The area on thesilica gel plate showing the band having an Rf value identical with thestandard immunomycin was scraped and eluted with ethyl acetate. Theethyl acetate extract was washed with water, dried under nitrogen andthe residue was subjected to the HPLC analysis on Partisil 5-ODS-3column with acetonitrile:water:phosphoric acid (65:35:0.1) as theeluting solvent. One milliliter fractions were collected and a portionof each fraction was examined for the radioactivity.

Isolation of the 31-O-methyltransferase (DIMT)

(All of the procedures were carried out at 4° C. in the cold room or onice). Washed mycelium was suspended in 50 mM phosphate buffer, pH 7.5containing 1 mM PMSF and 1 mg/ml of lysozyme. The resulting suspensionwas stirred overnight and centrifuged at 15000 rpm for 30 minutes. Thepellet thus obtained was sonicated for 2' at 30" intervals bymicrotip-equipped sonifier. The cell homogenate was centrifuged asbefore and the supernatants were combined and re-centrifuged at 105K× gfor 45 minutes. The supernatant was then brought to 1% streptomycinsulfate, stirred for 30 minutes and centrifruged at 105K × g as before.The resulting supernatant, designated as the crude extract, was made tocontain 0.1 mM S-adenosyl-L-methionine (SAM) and 10% ethanol followingthe buffer system suggested for the isolation of the methyltransferasesfrom Strepomyces fradiae, a tylosin-producing organism (See N. J.Bauern, et al., J.B.C. Vol. 263, D. 15619 (1988).) The crude extract wasthen brought to 30% ammonium sulfate saturation and after 30 minutesstirring was centrifuged at 15000 rpm for 30 minutes. The resultingsupernatant was brought to 60% ammonium sulfate saturation and afteridentical stirring and centrifugation procedures, its precipitate wasrecovered. This fraction, designated as 30-60% cut, was dialyzed against50 mM phosphate buffer, pH 7.5, containing 1 mM PMSF, 0.1 mM SAM and 10%ethanol (Buffer L) using PD-10 Pharmacia column. This fraction waschromatographed on a DEAE-Sepharose containing column. This column waseluted stepwise with buffer L containing various concentrations of KCl.The DIMT activity was completely eluted with the buffer containing 0.3MKCl. The DIMT active fraction was dialyzed against buffer L and appliedon the preparative MonoQ HR10/10-FPLC column previously equilibratedwith the same buffer. One milliliter fractions, eluted under a lineargradient from zero to 1M KCl with a 130 minute duration in buffer L witha flow rate of 60 ml/hr, were collected and examined for the DIMTactivity. Fractions, showing the DIMT activity were pooled and after thenecessary treatment was subjected to an analytical MonoQ HR5/5-FPLCcolumn. This column was developed as above except for the KCIconcentration which was 0.5M KCl and fractions were examined for theDIMT activity. The active fractions were pooled, and subjected tochromatofocusing. Chromatofocusing was carried out on Pharmacia FPLCsystem. Column was developed with the polybuffer and 1 ml fractions witha flow rate of 30 ml an hour were collected. The pH of the fractions wasadjusted to 7.2 immediately after elution and pH determination. Enzymeassay was carried out on various fractions and peak DIMT activity waslocated in fraction 35. This fraction was then applied on a Superose-12column. This column was eluted with buffer L containing 150 mM NaCl witha flow rate of 18 ml/hr. and 0.3 ml fractions were collected. Thefraction having the peak DIMT activity was finally subjected to a 12.5%native polyacrylamide electrophoresis (native-PAGE). Afterelectrophoresis, cuts were made across the face of the gel and each gelcut was eluted by diffusion and assayed for the DIMT activity. The DIMTactivity was located in the fraction eluted from the gel cut with Rfvalue higher than Rf value of the standard egg albumin in the same gel.

General Properties of DIMT

Examination of the optimal temperature, pH and metal requirement of theDIMT activity was carried out on the enzyme preparation obtained fromDEAE-Sepharose column. For kinetic analysis, a preparation which hadbeen purified through a second MonoQ column chromatography was utilized.

Molecular Weight and Isoelectric Point Determination of DIMT

The molecular weight of the native enzyme was determined by gelfilteration chromatography on Superose-12 column. Column wasequilibrated and run in buffer L containing 150 Mm NaCl and with a flowrate of 18 ml/hour. The apparant molecular weight of the denaturedenzyme was estimated by SDS-PAGE according to Laemmli (Nature (London)Vol. 227, pp. 680-685 (1970)). In both gel filteration and SDS-PAGE,bovine serum albumin (Mr=66000), egg albumin (Mr=45000), and carbonicanhydrase (Mr=31000), and cytochrome C (Mr=12400) were used asstandards. Isolectric point of the purified enzyme was estimated by bothchromatofocusing on a Pharmacia MonoP HR5/20 column with a pH intervalof 7-4 and calibrated isoelectric focusing-PAGE having interval of 3-9.

General Analytical Methods

Native-PAGE was carried out according to Laemmli, supra, with theelimination of the SDS from the buffer system. Protein concentration wasdetermined by the Bio-Rad assay system with bovine serum albumin asstandard.

Time Course of the Mycelial Growth and Immunomycin Production

Packed cell volume and immunomycin production, as a function of time,were determined as an indicator for the titer of the immunomycinbiosynthetic enzymes. Both the mycelial mass and immunomycin productionpeaked between 72 to 95 hours into the age of the cultures. It wastherefore decided to use 72 hour old mycelium for the isolation of theDIMT enzyme.

Properties and Localization of DIMT

Initially, using the crude extract, enzyme assay was established basedon the assay system previously reported for theS-adenosyl-L-methionine-dependent methylation of tylosin and avermectin(See Bauern, N. J. et al., J. Biol. Chem. 263, p. 15619 (1988) andSchulman, M. D. et al., Antimicrobial Agents Chemotherapeutics, 29, p.620 (1989). This preparation showed an absolute requirements for SAM andmagnesium ions and EDTA completely inhibited the activity. The enzymeactivity was also inhibited by ammonium sulfate, which was reversedafter complete dialysis. Optimal reaction temperature and pH were alsoestablished to be 35° C. abd 8.5, respectively. Using the optimalcondition, the enzyme activity could only be detected in the cell-freeextract (crude extract) obtained after the 105K× g centrifugation step.No enhancement of the activity was observed upon reconstitution of thepellet with the crude extract.

Isolation and Purification of DIMT

Using optimized assay condition, a 72-hour old mycelial cells providedus with a cell-free preparation which was functional with regard to DIMTactivity. The fraction containing this activity was isolated to a highdegree of purity after a series of purification steps which includedammonium sulfate precipitation, DEAE-Sepharose, preparative andanalytical MonoQ, chromatofocusing and Superose-12 columnchromatographies. The elution profiles and DIMT activity of thefractions from preparative MonoQ, analytical MonoQ, chromofocusing P andSuperose-12 columns were determined. The DIMT was consistently eluted ina single peak with increasing specific activity. High degree of purityof the DIMT was finally achieved when a highly purified fraction fromSuperose-12 column was subjected to native-PAGE. Fraction which waseluted from the native polyacryl-amide gel after electrophoresis wasenzymatically active and showed a single band on SDS PAGE andisoelectric focusing gel.

Molecular Weight and Isoelectric Focusing Point

Molecular weight of the purified DIMT was determined by SDS-PAGE andSuperose-12 column chromatography. From the calibrated SDS-PAGE thepurified DIMT showed a single homogeneous band with an apparantmolecular weight of about 32,000. This preparation was alsosymmetrically eluted from a calibrated Superose-12 column with anelution volume identical to that of a standard carbonic anhydrase(MR=31000), therefore indicating an apparent molecular weight of 31000for the native enzyme. Isoelectric point of the purified DIMT wasestimated by both chromatofocusing and calibrated isoelectricfocusing-PAGE. Both systems demonstrated an isoelectric point of 4.4 forthe native enzyme.

S-adenosyl-L-methionine (SAM) Dependency and Magnesium Ion Requirement

DIMT requires SAM and Mg⁺² for activity. As found, there is also majorinhibition of DIMT activity by EDTA, sinefungin andS-adenosyl-homocysteine (SAH), reconfirming the SAM and Mg⁺² requirementfor this enzyme.

pH and Temperature Dependencies of DIMT

The optimum activity of DIMT was seen at pH 8.5 and a temperature of 35°C.

Kinetic studies. With the established incubation conditions, the enzymicvelocity was directly proportional to the amount of protein up to about60 ug. Under similar condition, when 25 ug of the protein was incubatedfor different period of time, the initial velocity was maintained for 30minutes. Under the condition for measurement of the initial velocity,the apparent Km and Vmax of DIMT were determined with 40 uM SAM, whilethe kinetic utilization of SAM was examined with 25 umM of31-O-desmethylimmunomycin as cosubstrate. The apparent Km and Vmax ofDIMT for 31-O-desmethylimmunomycin and SAM were determined from theLineweaver-Burk reciprocal plots. The graphically obtained kineticparameters indicate values of 45.5 runole/hr/mg and 11 uM for apparentVmax and Km, respectively, when 31-O-desmethylimmunomycin was thevariable substrate. Similarly, values of 100 nmole/hr/mg and 12.5 uMwere estimated for Vmax and Km when SAM was utilized as the variablesubstrate.

EXAMPLE 3 General Procedure for Large-scale enzyme assay

The reaction mixture contained 35 mg of a partially purified DIMTfraction; 2 mM of magnesium sulfate; 6.25 uM substrate; 60 uM SAM in atotal volume of 10 ml which was adjusted with 50 mM of phosphate buffer,pH=7.30. The reaction was intiated by the addition of SAM underincubation conditions as previously reported. The reaction mixture wasextracted with ethylacetate and, after work-up, was subjected to a TLCsystem in which chloroform:methanol (9:1) was used as solvent. Thesilica gel from the area of the TLC plate, having Rf value that wasestablished from the isolation of radioactivity in a correspondingexperiment in which 14-C SAM had been used, was scraped off and elutedextensively with ethylacetate. Ethylacetate extract was then preparedfor purification by reverse-phase HPLC. Reverse-phase HPLC system wascarried out in a solvent system consisting ofwateriacetonitrile:phosphoric acid (45:55:0.1) at 60° C. Fractionseluting with an elution time corresponding to what had already beenestablished for the product of the enzymic assay in which 14-C SAM hadbeen used were collected and pooled. Pooled fractions were then furtherpurified for biological testing and chemical characterization.

Enzymatic Synthesis of the Compound II

Compound I was directly used in our radioisotope-based assay system asit was supplied. The retention time of this material was 19.44 minutesin our reverse phased HPLC system. The product of the enzymatic reactionin which this material and [³ H-CH₃ ]-SAM (S-adenosyl-L-methionine) hadbeen used as substrate/co-substrate was isolated and examined by theradio-TLC and radio-HPLC systems. In our radio-TLC, a radioactivefraction with characteristic Rf was detected which was suggestive ofmethylation of the substrate. On the radio-HPLC system the radioactivitywas eluted with fractions 25-27 with the peak elution of theradioactivity in fraction 26. Based on the informations obtained fromthe radio-TLC and radio-HPLC, a large scale enzymatic reaction wascarried out and the product of the reaction was isolated and examined byHPLC. The elution profile of this material with the peak elution time at25.15 minutes corresponding to fraction 26, similar to the elutionposition of the radioactive fraction. The elution time of thisenzymatically produced material is about four minutes shorter than theelution time (31.79 minutes) of the standard immunomycin in an identicalHPLC system, indicating distinct nature of this enzymatically producedcompound. This material was therefore isolated and submitted for FAB andNMR spectroscopic analysis. The result of FAB spectroscopy indicated theaddition of 14 atomic mass units [814-7(Li)=807] as a result ofenzymatic methylation and the NMR was characteristic (FIG. 2).

Biological Activity

HPLC purified samples of Compound I, Compound II and FR-900520 wereevaluated for biological activity in the same set of experiments. Theresults of these experiments indicated an IC₅₀ of about 11, 6, and 1 nMfor these compounds, respectively.

EXAMPLE 4 T-Cell Proliferation Assay

1. Sample Preparation

Purified Compound I, as prepared by HPLC above, was dissolved inabsolute ethanol at 1 mg/ml.

2. Assay

Spleens from C57B1/6 mice were taken under sterile conditions and gentlydissociated in ice-cold RPMI 1640 culture medium (GIBCO, Grand Island,N.Y.) supplemented with 10% heat-inactivated fetal calf serum (GIBCO).Cells were pelleted by centrifugation at 1500 rpm for 8 minutes.Contaminating red cells were removed by treating the pellet withammonium chloride lysing buffer (GIBCO) for 2 minutes at 4° C. Coldmedium was added and cells were again centrifuged at 1500 rpm for 8minutes. T lymphocytes were then isolated by separation of the cellsuspension on nylon wool columns as follows: Nylon wool columns wereprepared by packing approximately 4 grams of washed and dried nylon woolinto 20 ml plastic syringes. The columns were sterilized by autoclavingat 250° F. for 30 minutes. Nylon wool columns were wetted with warm (37°C.) culture medium and rinsed with the same medium. Washed spleen cellsresuspended in warm medium were slowly applied to the nylon wool. Thecolumns were then incubated in an upright position at 37° C. for 1 hour.Non-adherent T lymphocytes were eluted from the columns with warmculture medium and the cell suspensions were spun as above.

Purified T lymphocytes were resuspended at 2.5×10⁵ cells/ml in completeculture medium composed of RPMI 1640 medium with 10% heat-inactivatedfetal calf serum, 100 Mm glutamine, 1 mM sodium pyruvate, 2×10⁻⁵ M2-mercaptoethanol and 50 μg/ml gentamycin. Ionomycin was added at 250ng/ml and PMA at 10 ng/ml. The cell suspension was immediatelydistributed into 96 well flat-bottom microculture plates (Costar) at 200μl/well. The control, being the medium without test drug, and variousbelow-indicated dilutions of the sample (above-described purifiedCompound I) to be tested were then added in triplicate wells at 20μl/well. FR-900506 was used as a standard. The culture plates were thenincubated at 37° C. in a humidified atmosphere of 5% CO₂ -95% air for 44hours. The proliferation of T lymphocytes was assessed by measurement oftritiated thymidine incorporation. After 44 hours of culturing, thecells were pulse-labelled with 2 μCi/well of tritiated thymidine (NEN,Cambridge, Mass.). After another 4 hours of incubation, cultures wereharvested on glass fiber filters using a multiple sample harvester.Radioactivity of filter discs corresponding to individual wells wasmeasured by standard liquid scintillation counting methods(Betacounter). Mean counts per minute of replicate wells were calculatedand the results expressed as percent inhibition of tritiated thymidineuptake (proliferation) as follows: ##EQU1##

The results of % inhibition at various concentrations of Compound I arepresented in the following Table:

                  TABLE                                                           ______________________________________                                        Effects of Compound I on the proliferative response of                        Splenic T-cells stimulated with ionomycin + PMA                                            Concentration                                                                             Percent of                                           Sample       (nM)        Inhibition                                           ______________________________________                                                     315.2       98                                                                157.6       98                                                                78.8        95                                                                39.4        89                                                                19.7        65                                                                 9.9        39                                                                 4.9         0                                                                 2.5         0                                                   ______________________________________                                         Notes:                                                                        1. Mouse T cell cultures were pulsed with .sup.3 H thymidine for 4 hours      prior to harvesting at 48 hours.                                              2. Standard FR900506 (10 ng/ml) gave 99% inhibition.                          3. IC.sub.50 = 11.5 ng/ml = 14.5 nM, for Compound I, and generally in the     range of 10 to 18 × 10.sup.-9 molar.                                    4. Inhibition of TCell proliferation by Compound I was reversed by the        addition of 50 units/ml of IL2 (recombinant IL2) at the initiation of         culture.                                                                      5. Compound II also gave positive results in the above described Tcell        proliferation assay.                                                     

What is claimed is:
 1. An immunosuppressant compound having thestructure: ##STR4## wherein R is OH or CH₃ O.
 2. A pharmaceuticalcomposition useful for preventing transplantation rejection comprising atherapeutically effective amount of a compound of claim 1 in combinationwith a pharmaceutically acceptable, substantially non-toxic carrier orexcipient.